Objective: To explore the repair effect of tissue engineering for annulus fibrosus (AF) injury in stress-stimulation environment.
Methods: Non-adhesive fibrinogen (Fib) representing the repair with non-stress stimulation and adhesive hydrogel of fibrinogen, thrombin and genipin mixture (Fib-T-G) representing the repair with stress stimulation were prepared to repair the AF lesion. The relationship between adhesion and stress stimulation was studied in rheological measurements, tension tests and atomic force microscopy (AFM) experiments. The repair effect of stress stimulation was studied in designed acellular AF scaffold models with fissures and defects. The models were repaired by the two different hydrogels, then implanted subcutaneously and cultured for 21 d in rats. Histology and qPCR of COL1A1, COL2A1, aggrecan, RhoA, and ROCK of the tissue engineering of the interface were evaluated afterward. Moreover, the repair effect was also studied in an AF fissure model in caudal disc of rats by the two different hydrogels. Discs were harvested after 21 d, and the disc degeneration score and AF healing quality were evaluated by histology.
Result: In interfacial stress experiment, Fib-T-G hydrogel showed greater viscosity than Fib hydrogel (24.67 ± 1.007 vs 459333 ± 169205 mPa s). Representative force-displacement and sample modulus for each group demonstrate that Fib-T-G group significantly increased the interfacial stress level and enhanced the modulus of samples, compared with Fib group (P < 0.01). The Fib-T-G group could better bond the interface to resist the loading strain force with the broken point at 1.11 ± 0.10 N compared to the Fib group at 0.12 ± 0.08 N (P < 0.01). Focusing on the interfacial healing in acellular AF scaffold model, compared with Fib + MSCs group, the fissure and defect were connected closely in Fib-T-G + MSCs group (P < 0.01). Relative higher gene expression of COL2A1 and RhoA in Fib-T-G + MSCs group than Fib + MSCs group in AF fissure and AF defect model (P < 0.05). The immunohistochemistry staining showed more positive staining of COL2A1 and RhoA in Fib-T-G + MSCs group than in Fib + MSCs group in both AF fissure and AF defect models. The degree of disc degeneration was more severe in Fib + MSCs group than Fib-T-G + MSCs group in vivo experiment (11.80 ± 1.11 vs 7.00 ± 1.76, P < 0.01). The dorsal AF defect in Fib-T-G + MSCs group (0.02 ± 0.01 mm2) was significantly smaller than that (0.13 ± 0.05 mm2) in Fib + MSCs group (P < 0.05). Immunohistochemical staining showed more positive staining of COL2A1 and Aggrecan in Fib-T-G + MSCs group than in Fib + MSCs group.
Conclusion: Genipin crosslinked hydrogel can bond the interface of AF lesions and transfer strain force. Stress stimulation maintained by adhesive hydrogel promotes AF healing.
The translational potential of this article: We believe the effect of stress stimulation could be concluded through this study and provides more ideals in mechanical effects for further research, which is a key technique for repairing intervertebral disc in clinic. The adhesive hydrogel of Fib-T-G+MSCs has low toxicity and helps bond the interface of AF lesion and transfer strain force, having great potential in the repair of AF lesion.
Keywords: Annulus fibrosus; Genipin crosslinking; Hydrogel; Mesenchymal stromal cells (MSCs); RhoA/ROCK; Stress stimulation.
© 2023 The Authors.